Molecular Analysis of Polycyclic Aromatic Hydrocarbon Degradation by Mycobacteria

多环芳烃（PAH）是一类由两个或多个稠合的苯环以线性、角状和簇状排列组成的芳烃。 由于在环境中发现的许多芳烃具有天然热解来源，它们必须在整个进化时期与微生物接触。 因此，能够降解芳香族化合物的微生物的进化并不令人惊讶。 几乎所有关于PAH代谢的已知信息都是基于快速生长的革兰氏阴性生物和萘（二环）和菲（三环）作为模型底物。 生长缓慢的革兰氏阳性微生物，如分枝杆菌，已被证明不仅降解萘和菲，而且降解由四个或更多个芳环组成的PAH。 事实上，这类生物可能是最好的研究组，因为他们的能力，矿化高分子量多环芳烃的。 已经提出了基于在培养上清液中检测到的几个中间化合物的鉴定的分解代谢途径。 本研究将利用分子遗传学和生物化学的方法研究分枝杆菌PYO 1菌株降解高分子量多环芳烃的能力。 含有菲和芘降解所需的PYO 1基因的粘粒克隆已经被鉴定，并且迄今为止获得的30 kb序列的分析已经鉴定出至少21个参与PAH降解的基因，其与5个插入序列混合。 假定的功能已分配给每个开放阅读框，并将使用异源宿主中的表达、mRNA分析和敲除突变体进行生物学分析。 这些实验预计将导致分解代谢途径中的每个步骤的确定，并鉴定每个分解代谢中间体。 在环境中发现了高度挥发性的、稳定的多环芳香族化合物。 这种化合物可以在煤气化和液化过程中形成，不适当的废物焚烧做法，并在森林火灾。 这些化合物的来源包括石油、炭黑、煤焦油和其他石油衍生产品。分枝杆菌属细菌及其亲缘种在降解这些化合物的能力方面是独特的。然而，人们对这些能力的分子和生物化学基础知之甚少。 本项目将在基因和酶水平上研究分枝杆菌菌株降解多环化合物的能力。 彻底了解分枝杆菌如何降解这些化合物将有助于设计生物修复过程，从环境中去除多环化合物。

ZylstraPolycyclic aromatic hydrocarbons (PAH's) constitute a class of aromatic hydrocarbons consisting of two or more fused benzene rings in linear, angular, and cluster arrangements. Since many of the aromatic hydrocarbons found in the environment have a natural pyrolytic origin, they must have been in contact with microorganisms throughout evolutionary periods of time. It is not surprising, therefore, that microorganisms capable of degrading aromatic compounds have evolved. Almost all of what is known about the metabolism of PAH's is based on fast-growing gram negative organisms and naphthalene (two rings) and phenanthrene (three rings) as model substrates. Slow-growing gram positive organisms such as Mycobacterium have been shown not only to degrade naphthalene and phenanthrene but also to degrade PAH's consisting of four or more aromatic rings. In fact, this class of organisms is perhaps the best-studied group because of their ability to mineralize high molecular weight PAH's. Catabolic pathways have been proposed based on the identification of a few intermediate compounds detected in culture supernatants. In this project molecular genetics and biochemistry will be used to investigate the ability of Mycobacterium strain PYO1 to degrade high molecular weight PAH's. Cosmid clones containing the PYO1 genes required for phenanthrene and pyrene degradation have been identified, and analysis of the 30 kb of sequence obtained so far has identified at least 21 genes that are involved in the degradation of PAH's intermingled with five insertion sequences. Putative functions have been assigned to each open reading frame and will be tested with biological analyses using expression in heterologous hosts, mRNA analyses, and knock-out mutants. These experiments are expected to result in the determination of every step in the catabolic pathway with the identification of every catabolic intermediate. Highly recalcitrant, stable polycyclic aromatic compounds are found in the environment. Such compounds can be formed during coal gasification and liquification processes, improper waste incineration practices, and in forest fires. Sources of the compounds include oil, carbon black, coal tar, and other petroleum-derived products. Mycobacterium species of bacteria and their relatives are unique in their ability to degrade these compounds. However, little is known about the molecular and biochemical basis of these abilities. In this project, the ability of Mycobacterium strains to degrade polycyclic compounds will be investigated at the gene and enzyme levels. A thorough knowledge of how Mycobacteria degrade these compounds will aid in the design of bioremediative processes for removing polycyclic compounds from the environment.